Mycarosyl macrolide antibiotics

ABSTRACT

Covers the desisovaleryl derivative of niddamycin. Also covers a method of performing the microbial conversion of niddamycin, magnamycin A, magnamycin B, leucomycin A1 and leucomycin A3 to the corresponding desisovaleryl derivatives.

United States Patent 1 Theriault [451 Jan. 8, 1974 MYCAROSYL MACROLIDE ANTIBIOTICS [75] Inventor: Robert John Theriault, Kenosha,

Wis.

[73] Assignee: Abbott Laboratories, North Chicago, 111.

[22] Filed: Apr. 17, 1972 [21] Appl. No.: 246,095

[52] US. Cl. 195/81, 195/80 R, 260/210 AB [51] Int. Cl C12k l/00 [58] Field of Search 260/210 AB, 210 R;

[56] References Cited UNITED STATES PATENTS 2,796,379 6/1957 Tanner et al 195/80 R 3,630,846 12/1971 Hata et a1. 195/80 R Primary Examiner-A. Louis Monacell Assistant Examiner-Robert J. Warden Att0rneyRobert L. Niblack et a1.

[5 7 ABSTRACT cin B, leucomycin A and leucomycin A to the corresponding desisovaleryl derivatives.

6 Claims, No Drawings MYCAROSYL MACROLIDE ANTIBIOTICS BRIEF DESCRIPTION OF THE DISCLOSURE This invention relates to a new and useful derivative of niddamycin and to a method of preparing via micro bial conversion said niddamycin derivatives as well as derivatives of magnamycins A and B and leucomycins A, and A More particularly, this invention pertains to a niddamycin antibiotic composition having the following structural formula:

/C lS The invention is also particularly concerned with a method of preparing the following antibiotic compositions falling within the structural formula:

' Penicillum dalae NRRL2025 Penicillum species A-85 NRRL3282 Sporotrichum sulfurescens ATCC7 I 59 H CH0 cm on, l I I \N/ H 10 cm 11 l H 11 H 0 R1 H OCH: H H

-Rz RaO-H CH: H CH; 13 O CH: H H 0 "l 0 o-dcmcn \V O/ H I H OH CH l 3 .CH3 H \0 III II Where Z is Trichoderma viride NRRLI762 j 'b'j Pestalotiopsis royenae ATCCl 1816 A A Streptomyces (cinnamomeus) NRRLBI285 H Streptomyces factor NRRL31 13 R and R when taken separately are each hydrogen and when taken together are epoxy; and R represents hydrogen and acetyl.

These compounds are prepared by the microbial conversion of niddamycin, magnamycin A, magnamycin B, leucomycin A and leucomycin A to the corresponding desisovaleryl compounds falling within the structural formula just set out. The microbial conversion is carried out by inoculating one of the cultures listed below in a fermentation medium for sufficient time to allow incubation and growth, adding the niddamycin, leucomycin or magnamycin substrate to the fermentation medium, allowing sufficient time in order to permit the conversion to the corresponding desisovaleryl derivative to take place, and isolating said desisovaleryl derivative.

DETAILED DESCRIPTION OF THE INVENTION In more detail, the desisovaleryl derivatives of niddamycin, magnamycins A and B and leucomycins A, and A, are prepared by microbial transformation by resort to the following micro-organisms:

Streptomyces species Act-27 NRRL3948 Lentodium squamosum G-l7 NRRL3943 Dermoloma species F-27 NRRL3942 The notation ATTC indicates a culture of the organism has been placed on deposit with the American Type Culture Collection, Rockville, Maryland; the notation NRRL indicates the culture of the organism has been placed on deposit with the Northern Utilization Research and Development Division, Department of Agriculture, Peoria, Illinois; and the notation QM similarly indicates thqqssqs tsuyis.tlia uqtermas et fiesearch aiul fievelopment Center, United States Army, Natick, Massachusetts.

Suitable growth media for the micro-organisms comprises assimilable sources of carbon, nitrogen, defoamers and buffers. Examples of such nitrogen sources include soy bean flour, yeast extract, corn meal, oatmeal, meal extracts, distillers solubles, protein hydrolysates, peptones, amino acids, urea, nitrates, and amonium compounds. Carbohydrates, especially monosaccharides can be used as a. carbon source and include glucose, frutose, sucrose, maltose, lactose, molasses, dextrines, and starches.

The fermentation media used in this process are designated as A, B and C and contain the following ingredients:

Fermentation Media A Fermentation Media B g/liter Glucose monohydrate 50.0 (added post sterilization) Soybean flour 5 0 Yeast extract 5.0 NaCl 1.0 Malt extract 20.0 KH PO 4.1 K H P0 0.8

Adjust pH to 6.0 and add deionized water to 1.0 liter Fermentation Media C g/liter Glucose monohydratc (added post sterilization) Soybean grits Yeast extract NaCl K,HPO KH PO Adjust pH to 7.0 and add deionized water to 1.0 liter The following examples illustrate the process of the invention.

EXAMPLE 1 Cultures belonging to the genera Cunninghamella, Helicostylum, Mortierella, Monilia, Penicillum, Sporotrichum, Trichoderma, and Pestalotiopsis, were preferably inoculated from agar slant cultures into sterile cotton plugged flasks containing an increment of the sterile fermentation medium A. Those cultures belonging to the genus Streptomyces were preferably inoculated from agar slant cultures into similar sterile flasks containing an increment of sterile fermentation medium C. Cultures belonging to the genera Lentodium and Dermoloma were preferably inoculated from agar slant cultures into similar flasks containing an increment of sterile medium B. After inoculation, the flasks were incubated at a temperature of from 24-28 C. and preferably at approximately 28 C. on a rotary shaker. After 48 hours to 96 hours incubation, niddamycin was added to each flask at a level of 0.05 percent (50 mg/lOO ml. medium). The flasks were again incubated on the shaker. The flasks were sampled at various ages during the fermentation and analyzed by thin-layer chromatography in order to follow the rate of conversion.

The chromatographic analysis was carried out as follows:

wmilliliter s o f 'whdle eiiairwere adjusted To pI I of methanol. The methanol solutions were spotted (200 microliters on 20 20 cm. glass plates coated with Merck-Darmstadt silica gel plates. The thin-layer plates were developed in one of the following solvent systems:

I Cl-l Cl (:15:1)

ll CH CI 95 percent aqueous CH OH:NH OH (:10:l)

After 30 to 45 minutes developing time, the thinlayer plates were removed and dried. Ultra-violet absorbing compounds were photographed with ultraviolet light (254nm.) Niddamycin and related microbial conversion products were then revealed by spraying the plates with anisaldehyde reagent.

percent aqueous CH OH: H O

Niddamycin moved to an R of 0.65 to 0.70 in solvent system II and 0.77 to 0.82 in solvent system I. In solvent system 11 the product desisovalerylniddamycin had an R, value of 0.3 and 0.37. In solvent system I desisovalerylniddamycin had an R, value of from 0.57 to 0.65. All of the previously cited cultures formed varying amounts of the apparent desisovalerylniddamycin by thin-layer chromatography R,. This product absorbed short wave ultra-violet light at 254 nm indicating probably no change in the conjugated system. After spraying the plates with anisaldehyde reagent (95 percent C H OHzconc H 80 anisaldehyde (9:1:1) and heating to C. for 5-10 minutes in an oven, niddamycin was revealed as a black spot. The microbial conversion product was characterized by a rather unique purple-red color. Like niddamycin, the new antibiotic inhibited B. subtilis and S. aureus on bioautograph plates.

EXAMPLE 2 20 cotton plugged sterile 500 ml. Erlenmeyer flasks each containing 100 ml. of sterile medium A were each inoculated with one-half of an agar slant culture of Cunninghamella elegans QM6784. 20 similar flasks each containing 100 ml. of sterile medium C were inoculated with one-half of an agar slant culture of Streptomyces species Act-27 NRRL3948. A11 flasks were incubated on a Gump rotary shaker at 28 C. After 48 hours incubation, 0.05 percent niddamycin (50 mg/100 ml. medium) was added to each flask. The flasks were returned to the shaker and sampled at various ages during the fermentation for solvent extraction and thin-layer chromatography analysis as previously described. Although harvest can take place anywhere from 144 hours to 312 hours, in this example flasks were harvested at 216 hours. The contents of the 20 .flasks of each culture were pooled separately. The

pooled harvest whole culture was adjusted to pH about 8.5 with NH OH, adding a substantially equivalent volume of acetone with mixing. The whole culture: acetone beer of each culture was then extracted with 2 volumes of ethyl acetate twice. The ethyl acetate extracts were dried under vacuum. The residue obtained from each culture extract was reconstituted in a small volume of methylene chloride and chromatographed in a silica gel column. The major microbial conversion product which again appeared to be the same for both cultures, was separated from residual niddamycin and other impurities on the column by or with increased levels of methanol in methylene chloride. Final purification of the major microbial conversion product of each culture was achieved by preparative thin-layer chromatography. The purest column fractions were concentrated and streaked on a series of silica gel GF thin-layer chromatography plates which were developed in the CH CL :95 percent aqueous CH OH:- H O (85:l5:l) solvent system. The major conversion product was located with 254 nm light, and eluted in methanol. The product was dried, redissolved in ethyl acetate, filtered and finally dried. The isolated purified product from both Cunninghamella elegans QM6784 and Streptomyces species Act-27 NRRL3948 was submitted for mass spectra analysis. Both products showed a loss of the isovaleric acid from the molecule indicating a tentative structure of desisovalerylniddamycin. The product from Streptomyces species Act-27 NRRL3948 exhibited at 100 MHz nmr spectrum which was consistent with a structure of desisovalerylniddamycin.

EXAMPLE 3 200 sterile cotton plugged 500 ml. Erlenmeyer flasks each containing 100 ml. of sterile medium C were each inoculated with one-half of an agar slant culture of Streplomyces species Act-27 NRRL3948. The flasks were then incubated on a Gump rotary shaker (about 250 tpm) at 28 C. for 48 to 72 hours. At that time, the culutre of mycelia from each flaskswere pooled in sterile 5 gallon glass carboys. The pooled mycelia was allowed to settle and the supernate fermentation liquor was decanted and discarded. The pooled mycelia was then washed three times and resuspended at the original volume with sterile ().()l M pH 7.0, K HPO KH PO buffer. The washed mycelia was then redispensed into the original 500 ml. cotton plugged Erlenmeyer flasks and at the same volume of 100 ml. 0.05 percent niddamycin was added in powdered form (50 mg/IOO ml. washed mycelia) to each flask. The flasks were returned to the shaker for anywhere from 8 hours to 72 hours during which time good yields of desisovalerylniddamycin were formed. 48 hours was usually optimal for complete utilization of the substrate and good yields of the desired product.

The washed mycelia, phosphate buffer suspensio from each flask was again pooled. Since none, of the desisovalerylniddamycin was present in the mycelia, the pooled mycelia buffer suspension was filtered through heavy frame press filter paper, and the mycelia was discarded. One half volume of methanol was added to the filtrate to further precipitate finely suspended mycelium which was again removed by filtration. The methanolphosphate buffer filtrate containing virtually all of the desisovalerylniddamycin was then concentrated under vacuum from approximately 18 to t 19 liters down to l to 2 liters at a temperature of C. The concentrated filtrate was then adjusted to pH 7.5 with NH OH and one volume of methanol was added. This suspension was then extracted three timeswith methylene chloride. The methylene chloride extracts containing all of the desired product were evaporated to dryness under vacuum yielding approximately 6 gm. of impure desisovalerylniddamycin plus other impurities. This material was chromatographed on a 6 cm. diameter glass column packed with Merck Darmstadt silica gel GF and developed with a solvent system consisting of ethyl acetate: methanol: H O(90:l0:l). Desisovalerylniddamycin fractions were eluted. pooled and dried. These fractions were further purified follows by chromatography on a partition column: g

A 5.4 cm. diameter glass column was partially filled with the mobile or upper phase of the following solvent system: n-heptane:benzenezacetonezisopropyl alcohol; 0.01M phosphate buffer pH 7 (25:10:15:l0:25). The glass column containing the mobile phase was then packed with silica gel GF previously moistened with the immobile or lower phase of the solvent system (0.9 ml/gm of silica gel) to a height of about 52 cm. Desisovalerylniddamycin dried fractions were dissolved in a small volume of the mobile phase, added to the column, and developed with the mobile phase. Column fractions were monitored by thin-layer chromatography. Desisovalerylniddamycin crystallized directly in the column fraction tubes. The mass spectrum and nmr spectrum of the crystalline product was again consistent with a structure of desisovalerylniddamycin.

EXAMPLE 4 Microbial Conversion of Niddamycin to Desisovalerylniddamycin in 30 Liter Fermentors Fermentor medium: Fermentor charge volume: Antifoam:

Sterilization time:

lnoeulum:

NRRL3948. Fermentor lncuhation 28C. Temperature Fermentor agitation 410 rpm rate Fermentor air rate 0.6 liter/liter/min.

Two fermentors (30 liter) were inoculated and incubated as described above for 48 hours. At that time, 0.05 percent niddamycin was added in powdered form to each fermentor. incubation was continued for an additional 24 hours to 72 hours. Samples were taken after substrate addition for thin-layer chromatography analysis as previously described; Optimal conversion of niddamycin to desisovalerylniddamycin occurred from 24 hours to 120 hours after inoculation. The har vested fermentation beer was pooled, adjusted to pH 7.5 with NH Ol-l and one volume of methanol was added. The methanol: harvest culture suspension was then filtered through a frame press and the filtrate was concentrated to about 1 .0 liter with a vertical evaporator under vacuum at about 30 C. The concentrated filtered beer was again diluted with one volume of methanol, adjusted to 7.5 with Nl-LOH and finally extracted with 3 volumes of methylene chloride three times. The methylene chloride extracts were combined and evaporated to dryness under vacuum. The residue obtained, containing all of the impure desisovalerylniddamycin, was dissolved in a small volume of methylene chloride: methanol (:5) and then chromatographed on a 6.0 cm. diameter glass column slurry packed with silica gel (31 in methylene chloridezmethanol (95:5 Desisovalerylniddamycin fractions were eluted, pooled, dried under vacuum and rechromatographed on the previously described partition chromatography column. The column was developed with the mobile phase of the solvent system and fractions of reasonably pure desisovalerylniddamycin were collected. Desisovalerylniddamycin was allowed to crystallize directly in the column fractions or the fractions were pooled, dried under vacuum as pure amorphous 12 and Molecular ion (M 699.3829 (Calculated (C 1.00 absolute methanol) Melting Point Mass Spectrum Optical Rotation hour given The 100 MHz nmr spectrum was also consistent with a structure of desisovalerylniddamycin.

The same preparative techni ir'iftfihedabet/865i, be used to produce the desisovaleryl derivatives of magnamycins A and B and leucomycins A, and A The desisovalerylniddamycin compound was then tested for its anti-microbial spectrum. The in vitro activity of the desisovalerylniddamycin compound was tested as follows:

The microbial conversion product of niddamvcin (desisovalervlniddariivcin was submitted for antibacterial testing against fifteen organisms. The standard agar twofold dilution method was used in Brain Heart infusion Agar, m1./p1ate. lnoculum was a loopful of a 1:100 dilution of a 24 hour broth culture with the exception of the Staphylococci which used as undiluted culture. incubation was at 37 C for 24 hours. Niddamycin as used as the comparison control. Results are as follows with M.l.C. values (minimum inhibiting concentration) being given in mcg/ml figures.

It should be understood that in the above structures when R, and R, are taken together to represent epoxy, no double bond then exists between carbon numbers 1 claim:

.1, Amic qlzia cenxsmiaxrasth QQEEQPWHS a desisovaleryl derivative from a substrate selected from the group consisting of niddamycin, magnamycin A, magnamycin B, leucomycin A, and leucomycin A which comprises the steps of inoculating a culture selected from the group consisting of Cunninghamella elegans ATCC9245 Cunninglzamella elegans QM6784 Cunninghamella echinulata QM6782 Cunninghamella echinulata NRRLl 1498 Cunninghamella verticillata ATCC8983 Helicostylum puriforme ATCC8992 Mortierella ramanniana ATCC 1 1715 Monilia fructicola NRRL3945 Penicillum chrysogenum Wisconsin Q-l76 Penicillum lilacinum NRRL895 Penicillum janthinellum NRRL2016 Penicillum dalae NRRL2025 Penicillum species A- NRRL3282 Sporotrichum sulfurescens ATCC7159 Trichoderma viride NRRL1762 Pestalotiopsis royenae ATCC 1 1816 Streptomyces (cinnamomeus) NRRLB1285 Streptomyces factor NRRL31 13 Slreptomyces species Act-27 NRRL3948 Lentodium squamosum G-l7 NRRL3943 Dermoloma species F-27 NRRL3942 in a fermenta tion medium for sufficient time to allow incubation and growth, adding one of said substrates to the fermentation medium, allowing sufficient time in order to permit conversion to take place, and isolating the corresponding desisovaleryl derivative of said substrate.

2. The method of claim 1 wherein the desisovaleryl derivative of niddamycin is produced from niddamycin substrate.

3. The method of claim 1 wherein the desisovaleryl derivative of magnamycin A is produced from magnamycin A substrate.

4. The method of claim 1 wherein the desisovaleryl derivative of magnamycin B is produced from magnamycin B substrate.

5. The method of claim 1 wherein the desisovaleryl derivative of leucomycin A, is produced from leucomycin A, substrate.

6. The method of claim 1 wherein the desisovaleryl derivative leucomycin A, is produced from leucomycin A substrate. 

2. The method of claim 1 wherein the desisovaleryl derivative of niddamycin is produced from niddamycin substrate.
 3. The method of claim 1 wherein the desisovaleryl derivative of magnamycin A is produced from magnamycin A substrate.
 4. The method of claim 1 wherein the desisovaleryl derivative of magnamycin B is produced from magnamycin B substrate.
 5. The method of claim 1 wherein the desisovaleryl derivative of leucomycin A1 is produced from leucomycin A1 substrate.
 6. The method of claim 1 wherein the desisovaleryl derivative leucomycin A3 is produced from leucomycin A3 substrate. 